The Rise and Fall of TRP-N, an Ancient Family of Mechanogated Ion Channels, in Metazoa

Abstract

Mechanoreception, the sensing of mechanical forces, is an ancient means of orientation and communication and tightly linked to the evolution of motile animals. In flies, the transient-receptor-potential N protein (TRP-N) was found to be a cilia-associated mechanoreceptor. TRP-N belongs to a large and diverse family of ion channels. Its unusually long N-terminal repeat of 28 ankyrin domains presumably acts as the gating spring by which mechanical energy induces channel gating. We analyzed the evolutionary origins and possible diversification of TRP-N. Using a custom-made set of highly discriminative sequence profiles we scanned a representative set of metazoan genomes and subsequently corrected several gene models. We find that, contrary to other ion channel families, TRP-N is remarkably conserved in its domain arrangements and copy number (1) in all Bilateria except for amniotes, even in the wake of several whole-genome duplications. TRP-N is absent in Porifera but present in Ctenophora and Placozoa. Exceptional multiplications of TRP-N occurred in Cnidaria, independently along the Hydra and the Nematostella lineage. Molecular signals of subfunctionalization can be attributed to different mechanisms of activation of the gating spring. In Hydra this is further supported by in situ hybridization and immune staining, suggesting that at least three paralogs adapted to nematocyte discharge, which is key for predation and defense. We propose that these new candidate proteins help explain the sensory complexity of Cnidaria which has been previously observed but so far has lacked a molecular underpinning. Also, the ancient appearance of TRP-N supports a common origin of important components of the nervous systems in Ctenophores, Cnidaria, and Bilateria.

Keywords: Cnidaria; domain rearrangements; mechanosensation; nematocyst evolution; neurobiology; protein evolution.

Fig. 1.—

Phylogeny of TRP protein families (A) and distributions of ankyrin domains in TRP proteins (B): Numbers at nodes indicate bootstrap supports and size of the polygons scales to the size of the families across all used genomes (see Materials and Methods for details). The bar plots in (B) show the distribution of ankyrin domains in proteins of the respective TRP families. “All” refers to all proteins, including all TRP proteins, from GenBank with at least one ankyrin domain. The bar plots are based on data from GenBank only and do not include our manually corrected gene models.

Fig. 2.—

Phylogenetic distribution of transient receptor potential (TRP) families across Metazoa. The sizes of TRP subfamilies which were found using custom-made HMMs are listed at the tips of a phylogenetic tree for a representative set of metazoan genomes which were used (see Materials and Methods for a complete set of used genomes and supplementary fig. S4, Supplementary Material online, for corresponding phylogeny and occurrences of TRP-N). The tree topology is based on Philippe et al. (2011). Presumed events of WGDs are indicated by blue ellipses. Red frame encloses genomes in which TRP-N could be identified. Blue frame indicates TRP-N proteins which are activated through a “push,” mechanism (see text for explanations). Cross indicates the point at which the only bilaterian TRP-N copy has most likely been lost, that is, at the root of amniotes. TRP-N proteins with manually curated (in this study) gene models are in bold, and genes that were resequenced and PCR confirmed for this study are in red.

Fig. 3.—

Expression and localization of individual TRP-N molecules in Hydra magnipapillata. (A–F) In situ hybridizations. (A) In situ hybridization for TRP-N1 showing expression of transcripts in the nests of developing nematocytes. Scale bar = 100 µm. Inset shows a close up of TRP-N1 positive individual nematocyte nests in the body column of the same animal as in (A). Scale bar = 50 µm. (B) In situ hybridization for TRP-N2 showing expression of transcripts in developing nematocytes, Scale bar = 100 µm. (C–F) Immunostaining with TRP-N4 antibodies. (C) Overview of localization of TRP-N4 protein in tentacles and body column by antibody staining. Scale bar = 100 µm. (D) Localization of TRP-N4 in the nests of developing nematocysts in the body column. Scale bar = 20 µm. (E) Localization of TRP-N4 in tentacles where it surrounds individual mature nematocyst capsules. Scale bar = 10 µm. (F) Transmitted light view on nematocysts in (E) showing localization around various nematocysts. Scale bar = 10 µm. (G–M) Immunostaining of cnidocils with pan-TRP-N antibody. (G) A single nematocyte with desmoneme and stained cnidocil. Scale bar = 5 µm. (H) Staining of cnidocils in tentacle. Scale Bar = 10 µm. (I) Surface view of tentacle indicating spot-like staining in the basal part of cnidocils. Scale Bar = 10 µm. (J) Staining of isolated cnidocil showing a gradient toward the base of the cilium. Scale bar = 10 µm. (K) Costaining of TRP-N in the cnidocil (red) and of phalloidin in the stereocilia (green). Scale bar = 5 µm. (L) Same nematocyte as in (E), showing phalloidin staining of the stereocilia only. Scale bar = 5 µm. (M) Scanning electron microscope of tentacle surface with cnidocil. Scale bar = 2 µm.